Starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose consists of linear chains of α-1,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.
Sugars from starch, in the form of concentrated dextrose syrups, are currently produced by an enzyme catalyzed process involving: (1) liquefaction (or viscosity reduction) of solid starch with an α-amylase into dextrins having an average degree of polymerization of about 7-10, and (2) saccharification of the resulting liquefied starch (i.e. starch hydrolysate) with amyloglucosidase (also called glucoamylase or GA). The resulting syrup has a high glucose content. Much of the glucose syrup that is commercially produced is subsequently enzymatically isomerized to a dextrose/fructose mixture known as isosyrup.
α-amylases (EC 3.2.1.1) hydrolyze starch, glycogen, and related polysaccharides by cleaving internal α-1,4-glucosidic bonds at random. For a number of years, α-amylase enzymes have been used for a variety of different purposes, including starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing. These enzymes can also be used to remove starchy stains during dishwashing and laundry washing. Laundry and dish soils vary greatly in composition and therefore also in their ability to be removed and few amylases in the market place can be used for both laundry and dish applications.
Halomonas species are Gram-negative, moderately halophilic bacteria that grow optimally in media containing 3-15% NaCl, although most will grow over a very wide range of salinities. Some species are alkaliphilic. The only described amylase from Halomonas sp. is from Halomonas meridiana DSM5425 (Coronado, M. J. et al. (2000) FEMS Microbiology Letters 183:67-71). This α-amylase (i.e., amyH) has been cloned and a deduced amino acid sequence (AmyH) predicted (Coronado, M. J. et al., (2000) Microbiology 146:861-868). The nucleotide sequence for the amyH gene (AJ239061) and the predicted amino acid sequence for the AmyH polypeptide (CAB92963) are available in GenBank. The amino acid sequence of AmyH is also set forth in SEQ ID NO: 1. AmyH is predicted to be a polypeptide of 457 amino acids, including a signal sequence of 20 amino acids and a mature polypeptide sequence of 437 amino acids.
While AmyH appears to belong to be a family 13 glycosyl hydrolases, it has low sequence identity with its closest homologues, as shown in the following Table:
SequenceHomologidentityα-amylase, catalytic region (Herpetosiphon aurantiacus54%ATCC 23779)α-amylase (Stigmatella aurantiaca DW4/3-1)54%Glycosidase (Pseudoalteromonas tunicata D2)54%α-Amylase from Pseudoalteromonas haloplanctis52%